| Stevia rebaudiana Bertoni is a perennial herb of Asteraceae. Its leaves contain stevioside, the sweetness of sucrose250to300times, is a non-toxic, high sweetness, low calorie, no side effects is a sweet ingredients. As a new sugar crop, with high economic value.In this paper, the diploid stevia as experimental materials, for the establishment of stevia Regeneration System; the autotetraploid vitro induction and identification; Second, tetraploid stevia photosynthetic characteristics, physiological indicators, active ingredient contentDetermination; tetraploid stevia DNA methylation differences analysis done a preliminary study, the main results are as follows:1. Collected from different areas of stevia, stevia content by HPLC method, filtering out the high-yield high-quality materials and the establishment of a sterile micropropagation system. In this experiment, the robust buds as explants stevia sterile rapid propagation system, Stevia sterilized explants optimal mix of75%ethanol and sterilized30s,0.1%HgC12disinfection4min, mortality rates are low and pollution. established MS+1.0mg/L6-BA+0.5mg/L KT+NAA0.2mg/L complex medium stevia uncertainthe optimal bud proliferation of cultured medium, the proliferation of the number of adventitious buds up to6.9, and adventitious buds sprouting fast, neat, robust growth, dark green color.1/2MS+0.1Omg/LNAA+0.01mg/L IB A stevia plantlets most suitable rooting medium,7.48average rooting, rooting rate was100%as early rooting out the root neat, root length, stout pale white, strong and healthy. Stevia plantlets an optimal acclimatization time of5days, transplanting seedlings in the20day survival rate was90.77%.2. In order to establish stevia sterile micropropagation system on the basis of0.05%,0.10%and0.20%colchicine solution soak stevia adventitious buds induced stevia to autotetraploid, and through chromosomesthe identification and flow cytometry ways identified ploidy The results of this study showed that buds immersed in0.20colchicine solution for12h prior to culture induced as high as32.14%autotetraploid plants. The autotetraploid plants was larger than in diploid plants; autotetraploid plants possessed larger, thicker and deeper green leaves, and distorted appearances in comparison with diploid plants; The chromosome numbers of diploid cells was2n=2x=22, antotetraploid cells was2n=4x=44; Flow cytometry ploidy identification and diploid DNA relative content of100, antotetraploid DNA content of200.3. The diploid and autotetraploid of Stevia rebaudiana Bertoni were used in this research and to determine their photosynthetic characteristics, including phylliform index, photosynthetic pigment content, Physiology and biochemistry indexes (net photosynthetic rate, stomatal conductivity, intercellular CO2concentration, transpiration rat, SOD, POD, CAT, MDA), stevioside content. The result showed that:Leaf area, Leaf thickness, Stomatal Length and width of autotetraploid were significantly higher than that of diploid; photosynthetic pigment content of autotetraploid was much higher than that of diploid. The autotetraploid of Pn、Gs、Ci and Tr were significantly higher than that of diploid; The activities of SOD, POD, CAT of autotetraploid lines are also higher than those of diploid; while the MDA content of autotetraploid was less than diploid; autotetraploid stevioside content is4.45%higher than the diploid and rebaudiosideA is4.68%higher than the diploid.4. The methylation sensitive amplification polymorphism (MSAP) was carried out to study the levels and patterns of DNA methylation in different ploidies plants. The polymorphism of autotetraploid DNA methylation was46.10%, while the diploid was46.53%. In comparison with diploid, in the autotetraploid genomic DNA total methylated rate and full methylated rate were decreased, but hemimethylated rate was increased. In the autotetraploid genomic DNA methylation patterns,29.72%was changed, including12.8%in the rate of hypermethylation,11.8%in the rate of demethylation and5.12%in the rate of methine-methylattion. |
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